Immunofluorescence (IFA) is an important immunochemical technique that allows for the detection and analysis of a wide range of antigens and antibodies within a tissue or cellular section. It can also be described as a microscopic method that utilizes a fluorescence microscope to identify antibodies against specific antigens. Additionally, it serves as a standard virology method for detecting the presence of antibodies based on their specific ability to react with viral antigens expressed in infected cells, and can identify viral proteins expressed in cells through antigen-antibody reactions. This test is often used to confirm positive results obtained from ELISA (Enzyme-Linked Immunosorbent Assay) or MFIA® (Multiplexed Fluorometric ImmunoAssay). It is typically employed as a confirmatory test, as the sites of antibody-antigen reactions can be visualized in infected cells.
Depending on the scope of the test or the specific antibodies used, two methods are available: direct (primary) or indirect (secondary).
Direct Method (Primary)
Direct Immunofluorescence (DIF) is a technique used in the laboratory for diagnosing skin diseases, kidney disorders, and other organ systems. It is also known as direct fluorescent antibody testing or primary immunofluorescence. Primary or direct immunofluorescence employs a single antibody that is chemically linked to a fluorophore. The antibody recognizes and binds to the target molecule, and the attached fluorophore becomes detectable through microscopy.
This technique offers several advantages over the secondary (or indirect) protocol, as the antibody is directly linked to the fluorophore (chromophore). This reduces the number of steps in the staining process, making it faster. It can also lower background signal by avoiding some issues related to cross-reactivity or non-specificity of the antibody. However, since the number of fluorescent molecules that can attach to the primary antibody is limited, direct immunofluorescence is less sensitive than indirect immunofluorescence.
Indirect Method (Secondary)
Indirect or secondary immunofluorescence utilizes two antibodies. The primary antibody, which is unlabelled, specifically binds to the target molecule, while the secondary antibody, which carries a fluorophore, recognizes and binds to the primary antibody. Multiple secondary antibodies can attach to a single primary antibody, enhancing the signal through an increased number of fluorophore molecules at each antigen.
This protocol is more complex and time-consuming than the primary (or direct) protocol but allows for greater flexibility, as different types of secondary antibodies and detection techniques can be applied to a specific primary antibody. This method is widely used as a confirmatory assay in HIV diagnostics.
Initially, virus-infected cells are grown on sterile 12 mm glass slides. To fix the antigen, the cells are fixed with paraformaldehyde to prepare the proteins in the cells for antibody binding. The cells are permeabilized with an appropriate detergent, such as Triton X-100. A specific antibody (e.g., patient serum) is applied to the surface to identify the viral antigen. A secondary antibody, specific to the Fc region of the primary IgG antibody, is then applied.
Since a fluorescent dye is linked to the secondary antibody, the antigen can be observed under a fluorescence microscope. Intracellular replication of the viral antigen can be revealed using IFA. Furthermore, two or even three antigens can be easily visualized simultaneously using two to three separate antibodies.
Test results are reported as positive, negative, or inconclusive. Currently, IFA is used for research purposes rather than diagnostic purposes. However, in the early days when ELISA kits were not commercially available, IFA was employed diagnostically using patient serum as the primary antibody.
The effective application of this method involves several considerations, including the nature of the antigen, the specificity and sensitivity of the primary antibody, the properties of the fluorescent label, the permeabilization and fixation methods, and the fluorescence imaging of the cells. Although each protocol requires fine-tuning based on the type of cell, antibody, and antigen, there are common steps for nearly all applications.
Immunofluorescence can be performed on tissue sections, cultured cells, or individual cells fixed using various methods. This approach can utilize antibodies to analyze the distribution of proteins, glycoproteins, and other antigenic targets, including small biological and non-biological molecules.
Fluorescence samples can be analyzed using various types of fluorescence microscopy. The simplest type is epifluorescence microscopy. While confocal microscopy is widely used, newer designs of super-resolution microscopes, such as STED (Stimulated Emission Depletion) microscopes and others, enable nanoscale imaging with much higher resolution.
Advantages of Immunofluorescence (IFA)
This method is cost-effective. Additionally, the morphology and location of fluorescence can be assessed to differentiate specific reactions from non-specific ones.
Due to performance limitations, IFA is not a primary screening tool for serological tests. This method requires a fluorescence microscope. Interpretations can be subjective, and results are not quantitative, often limited to one antigen per slide. Non-specific fluorescence is common, and fluorescence intensity can vary. Like all serological tests, IFA does not detect the infectious organism; it only indicates a marker of past infection. Since animals do not produce antibodies, IFA is not suitable for use in immunocompromised animals.
Correct Antibody Selection: Choosing the appropriate primary and secondary antibodies is crucial. Antibodies should specifically bind to the target antigen and be of high quality.
Positive and Negative Controls: Utilizing positive and negative controls is essential to ensure the accuracy and reliability of results. These controls help detect specific antibody binding and eliminate background noise.
Care in Washing Steps: Washing steps should be performed carefully and thoroughly. Incomplete washing can lead to background noise and reduced accuracy of results.
Use of Appropriate Solutions: Utilizing suitable solutions for blocking, incubation, and washing is critical. High-quality solutions can enhance accuracy and reduce background noise.